The impressive successes of the ongoing Human Genome Project have included ever-accelerating timetables for elucidating the complete genomic sequence of various organisms. As the human genome sequence nears completion, the sequencing of other vertebrate genomes is taking center stage. The recent initiation of sequencing projects for widely studied model organisms (e.g., mouse, rat, and zebrafish) will provide a valuable resource for future experimentation and important insights into vertebrate biology. Less clear is the relative value of other candidate genomes being considered for systematic sequencing, especially with regard to their potential contribution to the annotation and interpretation of the human sequence. To begin investigating such issues, we are generating large blocks of orthologous sequence from multiple vertebrates for detailed comparative analyses. Specifically, the same targeted genomic regions from multiple vertebrate specie are being isolated in large-insert clones and then sequenced. Efficient methods for designing orthologous hybridization probes and isolating BAC clones from the different species have been developed and implemented. Following characterization by several mapping methods, tiling paths of BACs are then selected and systematically sequenced. In total, greater than 100 Mb of comparative sequencing data is being generated each year. The establishment of this comparative sequence resource should facilitate the development of new computational tools for multi-species sequence comparisons, provide insight about the appropriate degrees of sequencing finishing that should be pursued in the sequencing of other vertebrate species, and reveal the benefits of sequencing species from a range of evolutionary distances.